For many years researchers in the field of bioconjugate chemistry have needed well-defined ligation strategies that can be used for modification of biomolecules. Efficient bioconjugation strategies generally involve high levels of functional group tolerance, compatibility with water and other solvents, and efficient conversions (e.g., fast reaction times and high yields). Reactions that adhere to the principles of “click chemistry” are ideal candidates for bioconjugation applications. “Click” reactions are thermodynamically driven because the products have a highly favorable enthalpy of bonds. Several reactions can be classified as “click”, including copper-catalyzed Huisgen's dipolar cycloadditions of azides and terminal alkynes, addition of thiols to alkenes, addition of isothiocyanates to amines, and Diels-Alder cycloadditions. Importantly, because the starting materials for these reactions are relatively stable, in principle they could be introduced to a wide range of macromolecules and hybrid materials. Furthermore, these reactions do not generate by-products and operate on reasonable timescales, making them attractive for use in bioconjugation.
Thiol modification is an important tool in the chemical, biological, medical, and material sciences. As the only thiol-containing amino acid, cysteine is typically used for protein modification using thiol-based reactions. Despite the ubiquity of cysteine tagging, general chemical approaches do not exist for the site-specific modification of a single cysteine in the presence of other unprotected cysteines within the same peptide/protein chain (FIG. 1A). Development of a general, robust, and highly efficient method that allows single-site-specific cysteine modification would significantly expand the ability to modify biomolecules.